Study On Impact Of Therapeutic Hypothermia Upon Neurologic Function After Global Brain Ischemia/reperfusion Injury And Related Mechanism

Objective:Global cerebral ischemia-reperfusion injury is a common emergency in medicine.When encountering ROSC after cardiac arrest,less than 5%patients survive with good neurological outcome.Induced hypothermia is the only therapy clinically proven to increase survival and improve neurological outcome of patients resuscitated from cardiac arrest.Although the optimal temperature range（32 to 34°C）is well established,critical gaps in our fundamental knowledge regarding the optimal time of onset,duration,and rate of rewarming have left clinicians guessing how to best apply this clinically effective intervention.The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following brain ischemia reperfusion injury.However,the relative contribution of calpain isoforms to the various forms of injury has not been determined as available calpain inhibitors are not isoform-specific.And Liebetrau found delayed moderate hypothermia reduced calpain activity and breakdown of its substrate in experimental focal cerebral ischemia in rats.So the role of m-calpain andμ-calpain during the global ischemia reperfusion injury is need to find out.There is accumulating evidence that therapeutic hypothermia（TH）combined with magnesium sulfate may be more neuroprotective than hypothermia alone after brain ischemia.Magnesium is an N-methyld-aspartate receptor and voltage-dependent calcium channel blocker.Miles found that postischemic intravenous administration of magnesium sulfate could inhibit hippocampal CA1 neuronal death after transient global ischemia in rats.And Zhu demonstrated that intravenous administration of magnesium is only neuroprotective following transient global ischemia when present with post-ischemic mild hypothermia.So the research is carried out to justify and guide further testing of this combination therapy in a global cerebral ischemia model after cardiac arrest.The results of this study will provide new ideas and theoretical support for the treatment of the global cerebral ischemia reperfusion injury.Method:1.Establishment of global cerebral ischemia reperfusion rat model of return of spontaneous circulation（ROSC）after a 10-min asphyxial cardiac arrest.2.Block randomization to normothermia（37±1°C）or TH（33±1°C）initiated 0,1,4,or 8 hrs after ROSC and maintained for 24 or 48 hrs once return of spontaneous circulation（ROSC）achieved.3.Comparition of 7-day survival among groups with Kaplan-Meier analysis.4.Comparition the suvival with good neurologic function that scores≥450 across the groups with Chi-Square test.5.Surviving hippocampal CA1 pyramidal neurons quantification after immunofluorescent staining among different experimental groups with stereology system for 7-day survived rats.6.Knockdown expression of the targeted isoforms in adult rat hippocampal CA1 pyramidal neurons with adeno-associated viral vectors expressing short hairpin RNAs targeting the catalytic subunits ofμ-or m-calpain.7.After6-minute transient forebrain ischemia,comparition of hippocampal CA1 pyramidal neurons 72-hour and 2-week survival betweenμ-calpain and m-calpain knockdown group after immunofluorescent staining.8.Observation of synergistic neuroprotection with combined magnesium and hypothermia in a primary hippocampal neuron culture model of NMDA-mediated excitotoxicity.9.Comparition surviving hippocampal CA1pyramidal neurons quantification after immunofluorescent staining between intravenous magnesium combined with hypothermia and hypothermia alone in a rat model of ROSC after a 10-min asphyxial cardiac arrest.Results:1.TH initiated 0,1,4,and 8 hours after ROSC resulted in 7-day survival rates of 45%*,36%*,36%*,and 14%respectively compared to 17%for normothermic controls（*p<0.05 vs.normothermia）.2.There was no difference in survival when TH was maintained for 24 vs.48 hours.3.There was no difference in good neurologic function when TH was maintained for 24 vs.48 hours.4.TH initiated 0,1,4,and 8 hours after ROSC resulted in good neurologic function of 24%*,24%*,19%*,and 0%respectively compared to 2%for normothermic controls（*p<0.05 vs.normothermia）.5.Hippocampal CA1 pyramidal neuron counts were 53?27%*,53?19%*,51?24%*,and65?16%*of normal respectively when TH initiated 0,1,4,or 8 hrs after ROSC compared to 9%in normothermic controls（*p<0.05 vs.normothermia）.6.Counts were greater when TH was maintained for 48 hrs compared to 24 hrs（68%?15%vs.42%?22%,p<0.001）.7.Knockdown ofμ-calpain,but not m-calpain,prevented calpain activity72 hours and 2 weeks after 6-minute transient forebrain ischemia,increased long-term survival.8.In the primary hippocampal neuron culture model of NMDA-mediated excitotoxicity only the combination of high extracellular Mg²⁺（1.6 mM）and hypothermia（32?C）significantly increased survival.(*p<0.05 vs.other NMDA-treated samples.9.Hippocampal CA1 pyramidal neuron counts were 16%higher in rats treated with TH and intravenous Mg²⁺（89,333+/-17,008）compared to TH and intravenous NS（65,583+/-33,964）,but this difference was not statistically significant（p=0.34）.Conclusions:1.Therapeutic hypothermia initiated on 0,1 and 4 hours and last for 24 or48 hours can improve survival and neurologic function significantly after global brain ischemia.Therapeutic hypothermia initiated on 0,1,4 and 8 hours and last for 24 or 48hours can improve protect hippocampal CA1 neurons significantly after global brain ischemia,and is superior when hypothermia is maintained for 48 hours compared to 24hours.2.Treatment to hippocampal CA1 neurons with capn1 shRNA can reduce the calpain expression in vivo 72 hours after transient forebrain ischemia（TFI）and decrease neuronal death 2 weeks post-TFI.3.Hypothermia combined with magnesium treatment trends to protect the neurons after ischemia/reperfusion injury relative to hypothermia alone,but in vivo the difference is not statistically significant.